Invasion of surrounding brain tissue by isolated tumor cells represents one of the main obstacles to an effective therapy of glioblastoma multiforme (GBM). Gliomas encompass the majority of tumors originating in the central nervous system (CNS). In adults, the most common tumors are high-grade neoplasms derived from astrocytes or oligodendrocytes. The World Health Organization classifies these malignant tumors according to their degree of anaplasia into grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma) and grade IV (GBM)1.
Gliomas account for more than 50% of all brain tumors and are by far the most common primary brain tumors in adults. Despite, development of new diagnostic technologies, the survival rate is extremely low. Only 3% are still alive five years after diagnosis. The clinical outcome of malignant gliomas depends on the invasion of isolated tumor cells in the normal brain tissue. Migrating cells can escape the surgical ablation of the tumor and are then the prime targets of post-surgical radiotherapy and adjuvant chemotherapy. Chemotherapeutic agents and irradiation act primarily by inducing apoptosis. This induction of apoptosis often involves activation of the CD95 (Apo-1/Fas) death receptor/ligand system. Nevertheless, most malignant glioma cells are resistant to CD95-induced apoptosis. Here we show that triggering of CD95 increases migration/invasion in apoptosis-resistant human long-term and primary glioma cultures. That is, triggering CD95 may entail initiating CD95 activity by using an agonistic antibody to CD95 or recombinant CD95L.
The tendency of primary glioma tumors of migration over apoptosis increases with the degree of malignancy. CD95 mediates migration via the PI3K/ILK/GSK3-beta; /MMP pathway in a caspase-independent manner. Furthermore we tried to figure out the linker molecule downstream of CD95. A possible candidate was Phosphoprotein enriched in Diabetes/Phosphoprotein enriched in Astrocytes-15-kDalton” (PED/PEA-15). Knockdown experiments excluded PED/PEA-15 as linker molecule in the signaling pathway of migration mediated through CD95/CD95L-System. Most importantly, gamma-irradiation also increased migration of cells resistant to CD95-induced death. Irradiation-mediated migration could be blocked by neutralization of CD95L. Thus, a tumor's reaction to CD95 stimulation should dictate subsequent therapy options. See Kleber, S., “Gamma irradiation leads to CD95 dependent invasion in apoptosis resistant glioblastoma cells,” Ph.D. Thesis, Deutsches Krebsforschungszentrum, University of Heidelberg, Jan. 3, 2006 (urn:nbn:de:bsz:16-opus-59926), which is incorporated herein by reference in its entirety.
The main types of gliomas are ependymomas, astrocytomas, and oligodendrogliomas, although there also exist mixed cellular forms of glioma cell conditions, such as oligoastrocytomas.
In addition to a cellular characterization, gliomal tumors are also characterized according to pathology and the seriousness of cellular invasion, which is typically recognized by those in the field as a “grading” classification system.
The most commonly used grading system is the World Health Organization (WHO) grading system for astrocytomas. The WHO system assigns astrocytomas a grade from I to IV, with I being the least aggressive and IV being the most aggressive. Thus, pilocytic astrocytoma is an example of a WHO Grade I glioma; diffuse astrocytoma is an example of WHO Grade II; anaplastic (malignant) astrocytoma is an example of WHO Grade III; and glioblastoma multiforme is an example of WHO Grade IV. The latter is the most common glioma in adults and, unfortunately, has the worst prognosis for inflicted patients.
Generically, the “low grade” gliomas are typically well-differentiated, slower growing, biologically less aggressive, and portend a better prognosis for the patient; while the “high grade” gliomas, are undifferentiated or anaplastic, fast growing and can invade adjacent tissues, and carry a worse prognosis. High grade gliomas are highly vascular tumors and have a tendency to infiltrate tissues, create necrosis and hypoxia, and destroy the blood-brain barrier where the tumor is located.
There also are infratentorial gliomas, which occur mostly in children and supratentorial in adults. The infratentorial gliomas are located in all interior cerebral areas below the undersurface of the temporal and occipital lobes, extending to the upper cervical cord, and includes the cerebellum. The supratentorial region is located above the tentorium cerebelli and contains the forebrain.
Tumor grade is an important prognostic factor: median survival for grade III astrocytomas is 3 to 4 years and for grade IV astrocytomas 10 to 12 months. The most frequent glioma (65%) is the GBM1. Cellular resistance to multiple proapoptotic stimuli and invasion of migrating tumor cells into the normal surrounding brain tissue are the main obstacles to an effective therapy.
The current treatment of malignant gliomas (grade III and IV) involves surgery, followed by irradiation and chemotherapy. Chemotherapeutic agents and γ-irradiation act primarily by inducing apoptosis. Induction of apoptosis often involves activation of the CD95/CD95-ligand (CD95L; Apo1L/FasL) death system2,3. Binding of trimerized CD95L by the CD95 receptor leads to recruitment of the adapter protein FADD (Fas-associated death domain, MORT1)4 and caspase-8 and -10 into a death-inducing signaling complex (DISC)5. FADD contains a death domain (DD) and a death-effector domain (DED). Via its DD, FADD binds to the DD of CD956. The DED recruits the DED-containing procaspase-8 into the DISC7. Procaspase-8 at the DISC is activated through self-cleavage and commits the cell to apoptosis by activation of downstream effector caspases8.
The CD95/CD95L system is used by malignant glioma cells to increase their invasion capacity. CD95 induces cell invasion via the PI3K/ILK/GSK3β pathway and subsequent expression of metalloproteinases. Increased CD95L expression is exhibited by irradiated glioma cells that escape surgical ablation. Irradiation-induced CD95 activity increased glioma cell migration. In a murine syngenic model of intracranial GBM, CD95/CD95L expression was strikingly upregulated upon interaction with the surrounding brain stroma.
The degree of CD95 and CD95L expression positively correlates with the tumor grade. Here we show that triggering of CD95 caused apoptosis in less malignant tumors (WHO I-II) while the grade IV tumors were resistant to CD95-induced apoptosis. In these highly malignant cells, CD95 mediates migration/invasion. Binding of CD95 by the CD95L activates PI3K and ILK, thereby leading to inhibition of GSK3β and to the induction of metalloproteinases. Irradiation of apoptosis-resistant cells increased expression of CD95 and CD95L, which in turn increased metalloproteinase expression and subsequently, migration/invasion. In a syngenic mouse model of intracranial gliomas, CD95 and CD95L expression was upregulated upon interaction with the surrounding stroma. Neutralization of CD95 activity dramatically reduced the number of cells invading the contralateral hemisphere.